The long-term objective of this research is the development of rational methods for the design and improvement of membrane-penetrating, amphipathic peptides that are antibiotic or cytolytic, or can carry other drug molecules as cargo into cells. Critical knowledge in reaching this objective is the determination of the mechanism of membrane penetration by these peptides. Antimicrobial peptides are known to exhibit considerable target specificity, which appears to derive from the interaction of the peptides with the lipid bilayer of the target cell membrane without the intervention of protein receptors. Furthermore, the development of resistance by bacteria is much more difficult because it entails massive changes in the bacterial membranes. Cell-penetrating peptides (CPPs) have been shown to deliver drugs or nucleic acids into cells and their potential for the treatment of cancer is enormous. Polycationic CPPs appear to enter the cell via endocytosis; but how they leave the endosome remains poorly understood. Amphipathic CPPs are similar to antimicrobial peptides. They appear to cross the lipid bilayer, either the plasma membrane or the endosomal membrane, without the need for external energy. Our challenge, regarding both classes of peptides, is to understand the mechanism of translocation. Two main hypotheses concerning the mechanism of membrane permeation by these peptides are proposed and will be tested. To do that, a new set of assays to assess membrane translocation by these peptides has been designed. The prediction is that specific features of the peptide sequences are necessary for peptides to penetrate cells or disrupt the membrane. Surmounting cellular barriers, including intracellular compartments, is a major difficulty in the use of antibiotics, in drug delivery, and in molecular therapy. This proposal addresses one of the fundamental difficulties involves: the mechanism of membrane permeation. PUBLIC HEALTH RELEVANCE: This project seeks to establish principles for rational design of antibiotic and cell- penetrating peptides. Understanding how the primary structures of these peptides determine their ability to cross the lipid bilayer barrier of cell membranes is of paramount importance for the development of new antibiotics and molecular vehicles for targetted delivery of drugs or therapeutical agents into specific cells. Targetted delivery has vast potential clinical applications, for example in the treatment of cancer.